Structural Insight into the 14-3-3 Protein-dependent Inhibition of Protein Kinase ASK1 (Apoptosis Signal-regulating kinase 1)*

  1. Tomas Obsil‡,§2
  1. From the ‡Department of Physical and Macromolecular Chemistry, Faculty of Science, and
  2. ‖Department of Biochemistry, Faculty of Science, Charles University in Prague, 12843 Prague,
  3. §Institute of Physiology and
  4. the Institute of Microbiology, The Czech Academy of Sciences, 14220 Prague, and
  5. the **Institute of Physics, Faculty of Mathematics and Physics, Charles University in Prague, 12116 Prague, Czech Republic
  1. ↵1 To whom correspondence may be addressed. Tel.: 420-241062191; E-mail: veronika.obsilova{at}fgu.cas.cz.
  2. ↵2 To whom correspondence may be addressed. Tel.: 420-221951303; E-mail: obsil{at}natur.cuni.cz.

Abstract

Apoptosis signal-regulating kinase 1 (ASK1, also known as MAP3K5), a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family, regulates diverse physiological processes. The activity of ASK1 is triggered by various stress stimuli and is involved in the pathogenesis of cancer, neurodegeneration, inflammation, and diabetes. ASK1 forms a high molecular mass complex whose activity is, under non-stress conditions, suppressed through interaction with thioredoxin and the scaffolding protein 14-3-3. The 14-3-3 protein binds to the phosphorylated Ser-966 motif downstream of the ASK1 kinase domain. The role of 14-3-3 in the inhibition of ASK1 has yet to be elucidated. In this study we performed structural analysis of the complex between the ASK1 kinase domain phosphorylated at Ser-966 (pASK1-CD) and the 14-3-3ζ protein. Small angle x-ray scattering (SAXS) measurements and chemical cross-linking revealed that the pASK1-CD·14-3-3ζ complex is dynamic and conformationally heterogeneous. In addition, structural analysis coupled with the results of phosphorus NMR and time-resolved tryptophan fluorescence measurements suggest that 14-3-3ζ interacts with the kinase domain of ASK1 in close proximity to its active site, thus indicating this interaction might block its accessibility and/or affect its conformation.

Footnotes

  • ↵* This work was supported by the Czech Science Foundation (Project 14–10061S) and the Czech Academy of Sciences (Research Project RVO:67985823 of the Institute of Physiology). The authors declare that they have no conflicts of interest with the contents of this article.

  • ↵Graphic This article contains supplemental Figs. S1–S3 and Tables S1 and S2.

  • Received February 26, 2016.
  • Revision received July 9, 2016.
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  1. First Published on August 11, 2016 doi: 10.1074/jbc.M116.724310 The Journal of Biological Chemistry 291, 20753-20765.
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